We tried to identify downstream target genes of miR-1307-5p which promote extrahepatic metastasis. Target gene prediction using TargetScan 7.2 identified 120 candidates as miR-1307-5p target genes. (Figure 6A) Next, the expression of 120 genes were evaluated in TCGA_LIHC cohort. Because miRs negatively control their target genes, we tried to find significantly down regulated genes in HCC tissue. In TCGA_LIHC cohort, 16 of the 120 genes were down-regulated in HCC tissue compared to adjacent non-tumor tissue, and 9 of 16 genes, including ALDH8A1, C11orf96, CLYBL, EFNB3, ENG, NPC1L1, PIM3, SEC14L2, and SLC8A1, demonstrated statistical significance (P < 0.05).
(Figure 6A and Figure 7)
To verify inversely correlated genes with miR 1307-5p, Pearson’s correlation analysis was performed using the expression data in TCGA_LIHC database. Expression of 5 of 9 genes, including ALDH8A1, C11orf96, CLYBL, ENG, and SEC14L2, showed inverse correlation with miR-1307-5p expression with statistical significance (r=<-0.3 and P <
0.05). (Figure 6B) We performed a pathway analysis with a functional annotation of the EMT using the IPA software on miR-1307-5p and the 5 target candidate gene (Figure 6C and Figure 8). As a consequence, it was confirmed that miR-1307-5p/SEC14L2/AKT and miR-1307-5p/ENG signal pathways were linked to EMT promotion. Survival analyses according to expression of ENG and SEC14L2 were performed using expression data in
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TCGA_LIHC. Figure 6D and Figure 6E illustrates Kaplan-Meier plot of OS and DFS according to expression of ENG and SEC14L2, respectively. Low ENG expression group showed significantly poor OS (P=0.0002) and poor DFS survival (P=0.0026) compared to high expression group. In addition to ENG, low SEC14L2 group demonstrated poor OS (P=0.011) and DFS (P=0.003) compared to high expression group.
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Ⅳ. DISSCUSSION
A growing body of evidence indicates that exosomes transfer pro-metastatic molecules to recipient cells, thereby resulting pre-metastatic niche formation. (14, 15) The present study was performed under the assumption that the expression of specific exo-miRs would be increased in systemic circulation prior to extrahepatic metastasis and they could play as a driver of metastasis in patients with HCCs.
To confirm the assumption, we analyzed differentially expressed circulating exo-miR profiles between metastasis-free group and metastasis group during follow-up.
Among the 61 predominantly over-expressed plasma exo-miRs in the metastasis group, further selection of candidate miRs were performed by systematic integrative analyses with publicly available RNA-seq datasets. Consequently, miR-106b-5p, miR-1307-5p and miR-340-5p, were selected as potential candidates for pro-metastatic miRs. In validation study, metastasis group demonstrates significantly overexpressed circulating exo-miR-1307-5p. Furthermore, down regulation of SEC14L2 and ENG and promoting EMT were proposed as possible downstream pathway of miR-1307-5p in comprehensive bioinformatics analyses. In our knowledge, this is the first study which identified circulating exo-miR-1307-5p as a novel metastasis driver molecule and metastasis predictor in HCC patients.
Previous studies reported that exosomes enclose unique cargoes from parent cell and the exosomal cargoes have been highlighted as a promising biomarker in cancer
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biology. Recently, several studies reported circulating exo-miRs as potential diagnostic biomarker for early stage HCC. (20, 21) Moreover, studies have shown that the expression of an abnormally regulated exo-miRs can promote HCC progression and metastasis, by altering the genetic network. (22) Lin et al. reported that exosome-mediated miR delivery promotes HCC EMT and metastasis in cell line study. (23) However, few studies have sought to identify circulating exo-miR profile as metastasis predictor and metastasis driver in HCC. In the present study, circulating exo-miR-1307-5p was identified as a potential candidate of metastasis predictor and metastasis driving molecule in HCC patients. Previously, miR-1307 has been reported as onco-miR in diverse cancers and serum exo-miR-1307 has been reported as ovarian cancer biomarker. (24-26) In HCC, one recent study reported that miR-1307 promote HCC growth and metastasis by inhibiting DAB2 interacting protein in HCC. (27) Although the present study identified exo-miR-1307-5p as a potential candidate of metastasis driver, further mechanism study is necessary to identify how circulating exo-miR-1307-5p promotes HCC metastasis and whether it could be potential therapeutic target of HCC.
Presence of vascular invasion has been considered as an important hallmark of HCC invasiveness and poor prognosis. (28) It also has been reported as a principal predictive marker for extra-hepatic metastasis of HCC. (29) In the current study, all of the nine candidate miRs were significantly overexpressed in patients with metastasis as well as vascular invasion. Considering vascular invasion is closely associated with subsequent extrahepatic metastasis in patients with HCC, exo-miR-1307-5p might be
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predictive marker for extrahepatic metastasis and/or vascular invasion.
EMT is a process that loses the characteristics of epithelial cells and acquires the mesenchymal phenotype. (30) EMT plays a crucial role in promoting tumor invasiveness and metastasis by inducing the loss of cell to cell adhesion. (31) In the present study, we proposed SEC14L2/Akt and ENG-related signaling pathways as downstream pathway of miR-1307-5p for promoting EMT in HCC patients. According to pathway analysis using IPA, SEC14L2 is down-regulated by miR-1307, which in turn leads to Akt pathway activation, promoting EMT. SEC14L2 has been reported as a potent tumor suppressor gene in various malignancies. (32) In HCC, Li et al reported that SEC14L2, as a novel master regulator gene, exerted an anti-proliferative effect in HCC cells and strongly suppressed tumor growth in a mouse model. (33) ENG (CD105), a transmembrane glycoprotein, is one of the transforming growth factor β co-receptors. (34) ENG is involved in angiogenesis of solid tumors including HCC. (35) Several studies reported that down-regulation of ENG in HCC tissue, as well as serum concentrations, could play as a poor prognostic marker for HCC patients. (34, 36, 37) However, the molecular mechanisms of ENG in HCC progression are still poorly understood. According to the IPA analysis performed in this study, down regulation of ENG by miR-1307-5p promotes EMT by activating PLAU, RHOA, SMAD3, TWIST1 and MYC. Further mechanism research is needed to verify the downstream pathways proposed by the IPA.
This study has two limitations. The first limitation is the small number of the included patients. In our study, metastasis group was defined as patients who have
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developed extrahepatic metastasis after initial blood sampling point. Because HCC patients with the occurrence of metastasis who had available blood samples were rare, we could not enroll sufficient number of patients for powerful statistical analysis. From different points of view, due to the shortage of blood sample of metastatic HCC patients, we assume that the results of current study provide valuable and potentially useful information about pro-metastatic exo-miR. Further validation study in larger cohort is needed to verify the result of this study. Second, mechanism study of how exo-miR-1307-5p could facilitate extra-hepatic metastasis could not be performed. To overcome this limitation, we implemented in silico analytic strategy. MiR-1307-5p downstream pathway promoting EMT was predicted by using bioinformatics analyses. Experimental validation to verify the involved signaling pathway would be required as well.
In conclusion, our present study revealed circulating exo-miR-1307-5p as a predictive marker for the occurrence of metastasis in HCC patients using systematic integrative analyses. EMT promotion by down regulation of SEC14L2 and ENG could be possible downstream pathway of miR-1307-5p by comprehensive bioinformatics analyses.
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본 연구에서는 정상군 환자와 간암환자의 혈청과 혈장을 이용하였고, 간암환자 중에서도 전이가 있었던 환자와 전이가 없었던 환자를 분류하였다. 환자의 혈청과 혈 장에서 엑소좀을 추출하여 전사체 분석을 진행 하였고, 그 외에 GEO data와 TCGA data를 이용하여 간암 전이에 연관이 있는 엑소좀 microRNA 후보를 확인 하였다. 선 별된 엑소좀 microRNA 후보는 각 환자별로 실시간 중합효소 연쇄반응 실험을 통해 전 이와 밀접한 관련이 있는 엑소좀 microRNA를 선별하였다. 최종 선별된 엑소좀 miR-1307-5에 대하여 연관 있다고 예상되는 유전자를 확인 하였고 더 나아가 관련 유전자 와의 신호 전달 경로까지 확인한 결과 엑소좀 miR-1307-5p가 ENG를 막으면 여러 신 호 전달 경로를 통해 최종적으로는 EMT를 촉진한다고 예측되었다.
결과적으로 우리의 연구는 엑소좀 microRNA를 이용하여 간암 전이 관련 잠재 적인 바이오 마커로써의 가능성을 확인 하였고, 더 나아가 간암 전이 진단에 새로운 가능성을 기대한다.
핵심어 : 간세포암, 전이, 엑소좀, microRNA, bioinformatics 분석